Electrophoretic separation apparatus

ABSTRACT

An electrophoretic separation apparatus for the support-free continuous separation of molecular fractions in a buffer solution has a thin flat electrophoresis chamber defined between watercooled plates through which the mixture of fractions and buffer solution is passed and wherein the solution is subjected to a unidirectional field transverse to the direction of mass flow to separate the fractions. Subsequently the separated fractions are sedimented out of their respective portions of buffer solution in collecting vessels ahead of the outlets for the solution. Downstream of the collecting vessels is mechanical or hydrostatic pump or flow-control means which makes the fluid flow through the apparatus.

United States Patent Inventor Philipp Adolf Von Miinchhausen Grolmanstr.44-45, 1 Berlin, 12, Germany Appl. No. 808,046 Filed Mar. 13, 1969Patented Oct. 26, 1971 Priority May 10, 1968 Germany P 17 67 432.7

ELECTROPHORETIC SEPARATION APPARATUS 12 Claims, 5 Drawing Figs.

3,346,479 10/1967 Natelson 204/301 3,450,624 6/ 1969 Natelson 204/2993,519,549 7/1970 Grassmann et a]. 204/299 FOREIGN PATENTS 1,164,9843/1964 Germany 204/299 Primary ExaminerF. C. Edmundson Attorney-Karl F.Ross ABSTRACT: An electrophoretic separation apparatus for thesupport-free'continuous separation of molecular fractions in a buffersolution has a thin'flat electrophoresis chamber defined betweenwater-cooled plates through which the mixture of fractions and buffersolution is passed and wherein the solution is subjected to aunidirectional field transverse to the direction of mass flow toseparate the fractions. Subsequently the separated fractions aresedimented out of their respective portions of buffer solution incollecting vessels ahead of the outlets for the solution. Downstream ofthe collecting vessels is mechanical or hydrostatic pump or flow-controlmeans which makes the fluid flow through the apparatus.

PATENTED OCT 2 6 I97! SHEET 10F 4 Inventor Phil/pp Ado/f u MdnchhausenKm: 9

A fforney PATENTEUnm. 25 I971 sum '2 0F 4 Inventor:

Philipp Ado/f v. Miinchhausen by gza l JM.

Aftorn ey PATENTEDOBI 2s ism 3,616,455

SHEET 3 BF 4 Inventor:

Phil/pp Adolf vMUnchhausen by at g: 3C5

Attorney ELECTROPI-IORETIC SEPARATION APPARATUS My invention relates toan electrophoretic apparatus as used in the continuous separation ofmolecular fractions in a buffer solution.

BACKGROUND OF THE INVENTION The resolution of mixtures of electricallyseparable fractions, usually biological proteinaceous substances whichmay include whole cells, through electrophoretic processes, has becomeincreasingly widespread in recent years. Such processes recognize theprinciple that, when such a mixture of fractions is transported along amass-flow path in a conductive medium, a unidirectional electric fieldtransverse to the direction of mass flow produces a drift of the chargedparticles in the direction of the potential gradient, the effects ofelectrically induced migration on the different fractions are differentand that, therefore, the fractions drift to different extents.Collection of the fractions at a series of points along a line parallelto the electric potential gradient and transverse to the massflowdirection gives a clear resolution of the different fractions.

The devices hitherto proposed to carry out this process, aside fromthose using supports or carriers, generally have a series of collectingvessels arranged downstream of some sort of pump arrangement which isconnected to the actual separation or resolving chamber where thefractions are spread apart by the electric field. These pumpingarrangements inevitably damage the larger fragments, e.g. cells, whichcannot be recovered intact. This presents an enormous disadvantage ofthe devices when used in many biological separations.

OBJECTS OF THE INVENTION It is therefore the principal object of myinvention to overcome this disadvantage.

Another object is the provision of an improved thin-layerelectrophoresis method and apparatus which can be used for a largenumber of separation processes without damage to cells and the like.

SUMMARY OF THE INVENTION The collecting vessel or vessels of the instantinvention are provided between the pump and the output of the separationor electrophoresis chamber. This collecting vessel is advantageously aburette in which the fractions can sediment. The input opening andoutput openings of the burette are small (in cross section) in relationto its overall cross section so that the velocity of the buffer solutiontherein is sufficiently slow to permit this sedimentation.

The pump can be a noncontact peristaltic pump connected to the flexibletubing leading from the output opening of the burette. Since a pluralityof burettes will be used for the necessary resolution in the apparatus,this type of pump is advantageous in that it can be connected to aplurality of tubes or hoses and pump several burettes simultaneously.

In a further embodiment, a capillary tube is used as the pump orflow-control means. The supply of buffer (i.e. its reservoir) for thechamber input is mounted above this tube and a secondary reservoir canbe connected to it to form a liquid head in the tube.

According to this principal feature of my invention, the cells or otherfragile, bulky fractions are not injured during electrophoreticseparation.

A further feature of my invention is that the separation chamber isprovided with means enabling its rotation through 90", from a vertical(upright) to a horizontal (recumbent) position. This was previouslyconsidered impossible since it was through that the tilt would bringabout so-called streaking" but, surprisingly, no such effect is noticedwith the water-cooled construction of my invention.

The method of the present invention, therefore, involves the formationof mass-flow path for a buffer solution and the entraining a mixture offractions to be separated from an inlet side to an outlet side through athin flat chamber, while applying across this path a unidirectionalpotential gradient capable of separating the mixture of fractions intodiscrete portions which arrive at different locations along the outletside in the direction of the potential gradient; according to thisaspect of the invention, a continuous flow of the solution abstracted ateach location at the outlet side is induced through an upright elongatedsedimentation vessel adapted to retain at least temporarily a portion ofthe abstracted solution so that sedimentation of the separated fragmentsoccurs in this vessel. Intermittently, the vessel is drained at itsbottom between its inlet, which is connected to the electrophoresischamber, and an outlet connected at an upper location above the inletand the drain, to a flow control means of the character described. Theflow control means may include the noncontact pump mentioned earlier ora capillary through which the solution is driven by a hydrostatic head.

BRIEF DESCRIPTION OF THE FIGURES The foregoing and other features,objects and advantages of my invention will be described in greaterdetail hereinafter, reference being made to the accompanying drawing inwhich:

FIG. 1 is a side view of an electrophoresis apparatus according to mypresent invention;

FIG. 2 is an end view, partly in section, of the embodiment shown inFIG. 1;

FIG. 3 is a side \Yew, partly in section of a further embodiment of myinvention;

FIG. 4 is an expanded sectional detail of the chamber according to myinvention; and

FIG. 5 is a detail, partly in section, of FIG. I.

SPECIFIC DESCRIPTION FIG. I shows two watcr-cooled'plates 2 and 3defining a flat, narrow carrier-free unobstructed separation chamber Ibetween them. The plates and the chamber is mounted rotatably by a hinge31 on a support 34 so that an arm 21 (centered on the hinge and formedwith a slot 21a) allows tilting of the chamber upwardly through (seebroken lines in FIG. I) and securing the chamber by a locking nut 2Ib.

Attached to the support frame 34 carrying this hinge 31 is a rack inwhich a plurality of burettes 17 serving as the collecting means orvessels are held in clips (FIG. 2). These burettes 17 are connectedthrough output or discharge tubes or hoses 15 to output of thechamber 1. A valve 16, comprising two bars 16b and 160, can clamp theoutput hoses l5 shut by means of screws 16a spanning the bars. Theburettes I7 have upper stopcock valves 22 and lower stopcock valves I8;an outlet hose 32 made of rubber, silicone rubber, silicones orsynthetic resins lead through a variable stepless peristaltic pump 19 toa flask 33.

As seen in FIG. 5, the pump 19 consists of a housing 60 in which a shaft64 is joumaled. This shaft 64 mounts two disks 63 (only one shown)between which polyvinylchloridc rollers 61 and 62 are rotatablysupported. A motor 66 of stepless variable speed is connected through abelt 65 to this shaft 64. The tubes 32 from the outlet openings of aplurality of bulrettes I7 are led between these rollers 6I and 62 andthe housing 60 so that, owing to the cyclical compressing effect of therollers 61 and 62, liquid is pumped through the hoses 32 without beingtouched by the moving parts of the pump I9. A pump 19 is provided foreach rack of burettes 17, since one pump 19 can accommodate a pluralityof tubes 32.

FIG. 2 shows in particular the cooling and buffer-supply system of theapparatus. The interior of the plate 2 is divided by bars 6 to form azigzag path as shown by arrows 5 through which cooled water is pumped inat 4 and out at 4'. The plate 3 is similarly cooled. Two electrodetroughs I0 and I0, flanking the sides of the chamber and parallel to theflow direction, as shown in US. Pat. No. 3,305 ,47l of Feb. 2l, l96l,are mounted on opposite ends of the plate 2, with inlets I3 and I3 andoutlets l4 and 14'. An adjustable DC high-voltage source 47 is connectedacross terminals 12 and 12 of the electrodes l and The buffer solutionis fed in from a flask 26 through a valve 9. A manifold 8 supplies thiswlution to the whole upper edge (inlet side) ofthe chamber 1.

FIG. 4 best shows the construction of the chamber 1. Two plates 2a and2b forming the plate 2 and separated by a gasket 48 and the bars 6, andtwo further plates 3a and 3b separated by a gasket 49 and furtherspacers 6 provide means for cooling the chamber 1. The inlets 4 andoutlets 4 are also seen here.

The electrode troughs l0 and 10' are filled with a solution similar tothe buffer solution in the chamber 1, but approximately twice as strong,which is renewed or replenished as described below. In the bases ofthese troughs l0 and 10 are wads of packing 100 as described in theabove-cited patent which serve to permit electrical contact with thebuffer solution in the chamber 1.

The sheets 3b and 2b are separated by a gasket 50 and form theseparation chamber 1 whose inlets 8 and outlets are shown extendingthrough the gasket 50.

FIG. 3 shows how the connections 4 and 4' are joined to a source ofcoolant, in this case water, in a reservoir 46. This reservoir 46 isequipped with a cooling coil 45 of a refrigeration unit 44. A pump 46acirculates the water through the plates and the cooler. This coolingsystem tends to keep down the high temperatures generated by myapparatus.

Furthermore FIG. 3 shows a source 40 of double-strength buffer solutionconnected through a pump 41 to J-arms 43 which drip this solution intotwo cups 42 (only one shown) associated with respective electrodes 10and 10'. The outlets 14 (only one visible) drip into a waste flask 39.The necessity of dripping the solution rises from the fact that simplyconveying it through tubes would create a shunt across the electrodes 10and 10' greatly reducing their efflciency.

The sample to be treated is fed into the chamber 1 through a conduit 7(FIG. 4) from a flask 38.

The methods of feeding the coolant for the plates 2 and 3, the sampleinto the chamber 1, and the solution for the electrodes 10 and 10' shownin FIG. 3 are the same as used for the embodiment shown in FIGS. 1 and2.

FIG. 3 shows burettes or capillary tubes 17' which are mounted betweensupports 28 and 29 on a frame 27 in a plexiglass housing 30 and areconnected as described before to outlet tubes 15 of the chamber 1. Threevalves l6, l8 and 53 each consisting of a rod 24 serving to crimp aportion of tubing in a holder 52. This rod 24 rides on a bar 37 formedwith a bore 38 in which a handle 36 on the rod 24 engages. A spring 23mounted on a screw 23a on a support 230 on the bar 37 and on a screw 23bon the rod urges the rod 24 toward the holder 52. On depression of thebar 37, the handle 36 disengages the hole 38 and this spring 23 pulls inthe rod 24 thereby crimping the portion of tubing. Withdrawal of thehandle 36 such that it reengages in the hole 38, resets the valve formedthereby. The pinch-clamp valves 16 and 18 have the same function as thelike numbered valves in FIGS. 1 and 2 and are each provided for one tube15 or burette 17, respectively.

In addition, the valves 53 are provided between a reservoir 25 locatedabove the capillary tubes 17 and connections on the tops of these tubes17. Outputs of these tubes 17 are shown at 29'.

The difference in height between the reservoirs 25 and 26 forms ahydrostatic head across the tubes 17' which functions to control thepumping action.

In order to use my apparatus the pumps 41 and 46a are started and thehigh-voltage source 47 is turned on. A sample is fed in 7 and a shorttime is preferably waited for some spreading of this to take place underthe effect of the voltage. Thereupon the valves 9 and 16 are opened andthe pumps 19 are started (FIGS. l and 2) or the valves 53 are opened(FIG. 3). Periodically the valves 8 are opened to sample or recover thesediment in the burettes 17 or [7' and the cups are replaced beforeresampling.

The improvement described and illustrated is believed to admit of manymodifications within the ability of persons skilled in the art, all suchmodifications being considered within the spirit and scope of theinvention except as limited by the appended claims.

What is claimed is:

I. An apparatus for the continuous electrophoretic separation ofmolecular fractions in a buffer solution, said apparatus comprising:

an electrophoresis chamber having an inlet permitting ad mission of saidsolution into said chamber and an outlet permitting withdrawal of saidbuffer therefrom, thereby forming a continuous fluid flow through saidchamber;

electrode means in contact with said buffer for creating aunidirectional electric field across said flow;

pump means operatively connected with said outlet for drawing saidsolution therethrough out of said chamber; and

collecting means between said pump means and said outlet for sedimentingat least one of said fractions.

2. The apparatus defined in claim 1, further comprising conduitsconnecting said collecting means to said pump means and to said chamber,said collecting means including a collecting vessel having across-sectional area substantially greater than the cross-sectionaldiameter of said conduits to permit sedimentation in said vessel.

3. The apparatus defined in claim 1, further comprising a pair of spacedparallel plates forming said chamber between them and cooling means forpreventing overheating of said plates, said outlet comprising aplurality of outlet openings spaced along a wall of said chamber, saidinlet being formed along an opposite wall of said chamber.

4. The apparatus defined in claim 3, further comprising means fortilting said plates jointly through substantially from a substantiallyvertical position to a substantially horizontal position.

5. The apparatus defined in claim 1 wherein said collecting meanscomprises a substantially vertical burette connected at a lower endthereof to said outlet, and means connecting said burette to said pumpmeans at an upper end of said burette, said burette being provided atsaid lower end with means for draining off said one of said sedimentedfractions.

6. The apparatus defined in claim 1 wherein said pump means comprises aperistaltic pump, said apparatus further comprising a flexible conduitattached to said collecting means, said pump coacting with said flexibleconduit.

7 The apparatus defined in claim 1 wherein said collecting meanscomprises a capillary tube, said pump means comprising means formaintaining a head in said tube.

8. The apparatus defined in claim 1, further comprising valve meansbetween said outlet and said collecting means for selectivelyprohibiting fluid flow therebetween.

9. An apparatus for the continuous electrophoretic separation ofmolecular fractions in a buffer solution, said apparatus comprising:

a support;

a pair of spaced-apart liquid-cooled plates mounted on said support anddefining between them a narrow flat separation chamber having an inletside and an outlet side opposite one another whereby a mass flow of thebufl'er solution and mixture of fractions traverse said chamber fromsaid inlet side to said outlet side;

means for applying a generally unidirectional field across the mass flowthrough said chamber to separate said f ractions such that they arriveat different locations along said outlet side;

means at said outlet side for abstracting said fractions from saidchamber at said locations; and

tilting means between said support and said plates enabling tilting ofsaid chamber about a substantially horizontal axis through an angle ofabout 90, and for retaining said cham her in said tilted position.

10. The apparatus defined in claim 9 wherein said plates and saidchamber are generally rectangular and said sides are horizontal, saidtilting means including a hinge connected to said plates at said outletside of said chamber enabling swinging of said chamber between arecumbent position and an upright position ll. The apparatus defined inclaim 10 wherein said means for abstracting said fractions from saidchamber at said outlet side includes:

a respective flexible tube leading from said chamber and communicatingtherewith at each of said locations;

a respective upright elongated sedimentation vessel connected with eachof said tubes and communicating therewith at a location above the bottomof the respective vessel;

means at the bottom of each of said vessels for draining same;

means at an upper portion of each of said vessels above the location atwhich the respective tube communicates therewith for inducing a flow ofsaid solution through the corresponding tube and vessel whereby saidfractions are sedimented at the bottom of said vessels; and

means for selectively closing said tubes.

12. The apparatus defined in claim 11 wherein said vessels are buretteshaving valves at their bottoms, and said means for selectively closingsaid tubes are pinch clamps.

12. The apparatus defined in claim 11 wherein said vessels are buretteshaving valves at their bottoms, and said means for selectively closingsaid tubes are pinch clamps. 237] 6790103 F i i

2. The apparatus defined in claim 1, further comprising conduits connecting said collecting means to said pump means and to said chamber, said collecting means including a collecting vessel having a cross-sectional area substantially greater than the cross-sectional diameter of said conduits to permit sedimentation in said vessel.
 3. The apparatus defined in claim 1, further comprising a pair of spaced parallel plates forming said chamber between them and cooling means for preventing overheating of said plates, said outlet comprising a plurality of outlet openings spaced along a wall of said chamber, said inlet being formed along an opposite wall of said chamber.
 4. The apparatus defined in claim 3, further comprising means for tilting said plates jointly through substantially 90* from a substantially vertical position to a substantially horizontal position.
 5. The apparatus defined in claim 1 wherein said collecting means comprises a substantially vertical burette connected at a lower end thereof to said outlet, and means connecting said burette to said pump means at an upper end of said burette, said burette being provided at said lower end with means for draining off said one of said sedimented fractions.
 6. The apparatus defined in claim 1 wherein said pump means comprises a peristaltic pump, said apparatus further comprising a flexible conduit attached to said collecting means, said pump coacting with said flexible conduit.
 7. The apparatus defined in claim 1 wherein said collecting means comprises a capillary tube, said pump means comprising means for maiNtaining a head in said tube.
 8. The apparatus defined in claim 1, further comprising valve means between said outlet and said collecting means for selectively prohibiting fluid flow therebetween.
 9. An apparatus for the continuous electrophoretic separation of molecular fractions in a buffer solution, said apparatus comprising: a support; a pair of spaced-apart liquid-cooled plates mounted on said support and defining between them a narrow flat separation chamber having an inlet side and an outlet side opposite one another whereby a mass flow of the buffer solution and mixture of fractions traverse said chamber from said inlet side to said outlet side; means for applying a generally unidirectional field across the mass flow through said chamber to separate said fractions such that they arrive at different locations along said outlet side; means at said outlet side for abstracting said fractions from said chamber at said locations; and tilting means between said support and said plates enabling tilting of said chamber about a substantially horizontal axis through an angle of about 90*, and for retaining said chamber in said tilted position.
 10. The apparatus defined in claim 9 wherein said plates and said chamber are generally rectangular and said sides are horizontal, said tilting means including a hinge connected to said plates at said outlet side of said chamber enabling swinging of said chamber between a recumbent position and an upright position.
 11. The apparatus defined in claim 10 wherein said means for abstracting said fractions from said chamber at said outlet side includes: a respective flexible tube leading from said chamber and communicating therewith at each of said locations; a respective upright elongated sedimentation vessel connected with each of said tubes and communicating therewith at a location above the bottom of the respective vessel; means at the bottom of each of said vessels for draining same; means at an upper portion of each of said vessels above the location at which the respective tube communicates therewith for inducing a flow of said solution through the corresponding tube and vessel whereby said fractions are sedimented at the bottom of said vessels; and means for selectively closing said tubes.
 12. The apparatus defined in claim 11 wherein said vessels are burettes having valves at their bottoms, and said means for selectively closing said tubes are pinch clamps. 